The invention relates to bioassimilable boron compounds for fighting subviral particles which cause subacute, degenerative, non-inflammatory diseases and which are known as viroids.
Viroids are infectious particles which autonomously replicate in adequate host cells. They represent the smallest form of life. With subviral dimensions of about 10.times.1 nanometers, the rod-shaped structures are smaller than the most primitive virus. They consist of an annular molecule of ribonucleic acid; 246 to 375 nucleotides are combined, in a single strand and covalently closed to a tertiary structure of high stability wherein 2/3 of the opposite bases are paired in a complimentary fashion. There are also forms with open chains, simple and polymeric forms. Viroids are not surrounded by their own viroid-specific protein envelope. Up to now, viroids could be isolated as particles only from the cells of higher plants, but infectious ribonucleic acid cells of the same type have been found in connection with certain diseases also in the cells of vertebrates.
Besides the absence of cellular structures, a property which they share with the viruses, these smallest parasites are characterized in that they do not code any proteins. They do not leave any fingerprint in the protein machinery of the invaded cell, and in this manner, escape the antiviral defense mechanism of the infected host organism, at least during the incubation period.
In plants, the viroids cause no active or passive resistance. They induce neither hyperergic reactions nor the production of antiviral proteins.
The incubation periods are longer than with virus infections by orders of magnitudes. With long lived plants, they may be many years, but high temperatures (tropical climates) result in dramatic reductions of the latent periods.
The most frequent symptoms of a viroid infection in plants are:
growth stunts PA1 upsetting PA1 necrosis PA1 bearing of small fruits PA1 death of the plant. PA1 alienation of functions PA1 impediment of transmembrane transports PA1 blocking of disintegration PA1 protein polymorphism as a result of erroneous processing. PA1 1. Virosoids are well defined partially sequenced particles which contain a non-coding viroid component and which become infectious with the aid of a helper virus. They occur in plants (satellite viruses, multiparticle viral genomes) and in animals. PA1 2. Non-conventional Lenti-viruses represent an extensive still little defined group. Included therein are the pathogens of degenerative diseases of the central nervous system which always result in death after the symptoms appear; they have the above mentioned pathological and histochemical effects: spongelike perforation of the brain and nerve tissues by the death of nerve cells, formation of fibrilious and amyloidal protein aggregates by erroneous processing of genetic products, astrocytosis (hypertrophy of the astrocytes under formation of acid, fibrilious glycoproteins), behavioral disorders, progressive pathogenesis of numerous organs. The group of spongiform encephalitis includes: Scrapie diseases of sheep (for example, 18000 cases in one immunization accident in the UK) cattle madness (Bovine spongiform encephalitis=BSE, 10000 cases annually in GB) and, in humans, the Creutzfeldt-Jakob disease(CTE), the Gerstmann-Straussler syndrome (GSS), the Kuru-and the Alper's disease. The pathogens overcome the species barrier and are potentially infectious to all mammals. PA1 3. Homologies of the viroids with retroviruses and cell transforming DNA viruses. PA1 4. Subacutely persisting classical viruses. A number of degenerative diseases is caused by classical RNA viruses. After subsidence of the main infection, viral subspecies survive the immune defense in a defective condition with altered antigen pattern; they persist in the nerve strands and move there, similar to the Lyssa-viruses (rabies), from cell to cell. After, at times, very long latent periods, they cause grave, generally deadly neuropathological late follow-up diseases (measles, rubella, polio, or multiple sclerosis, the multiple sclerosis as a late follow up infection of non permissive human cells by Theilers Murines polio viruses). PA1 1) Viroids are highly complex double strand RNAs. Such segments serve, on one hand, as induction signals for the production of interferon which may result in the more or less complete neutralization of conventional viruses during the acute initial infection stage. If this mechanism does not work perfectly, classical viruses remain, leading potentially to late follow-up infections. PA1 5. Viroid participation on genetic diseases. In the area of plants, the egg cell is the main aim and most important vector of a viroidal infection. In analogy therewith, vertical viroid transmission with chromosomal damages to the egg cell cannot be excluded in the animal area. For genetic diseases which depend on the maternal part and which, and consequently correlate with the conception age thereof (for example Down Syndrome), a viroid participation in initiating the chromosome anomaly is well possible. An indication herefor is the brain amyloidosis, which may occur during the trisomy disease proceedings and which is otherwise characteristic for nonconventional, atypical "Slow Virus" diseases. PA1 the quasi indestructibility of the pathogens (classical hygiene and enzyme treatments are ineffective). PA1 The universal transmission routes (The importance of the transmission method is limited to influencing the duration of the symptom-free latent period: for example, massive seral inoculation close to the brain have the fastest results as it has been described for rabies whereas small amounts of infectious material administered orally resulted in extremely long incubation periods). PA1 non-existence of adequate safety tests (the death of the patient is the sole criterium). PA1 during surgical and dental treatments (surgical instruments, implants, electrodes) in dental nerves concentrated pathogens for example have led to the contamination of dentists and their patients. PA1 with all organs, tissue and bone marrow transplants. Particularly risky are organs after a certain time after the death of the donor when they are aged but still usable such as cornea, dura mater; the virulency stimulation of the pathogens is believed to be the cause by producing an increased amount of stress protein in the dying donor organisms. This means that the later the contaminated tissue is taken from the dying donor, the more infectious it is. PA1 with transfusions and serum products (a safe sterilization or decontamination of the products is impossible because of the small size and the high resistance of the pathogens). PA1 with extracts from human and animal organisms (hormones, interferon), the use of growth hormones of hypophyses of dead donors for example has led to the contamination of all the children age 4 to 16 treated with a particular sample. PA1 a risk exists also for all pharmaceutical strengthening and regeneration preparations which contain extracts of lipidrich animal organs.
The symptoms are qualitatively about the same for all types of plants and for all viroids which indicates a disturbance at a central location in the cell control of the host. Injected in mammals, the viroids do not cause an immune response, in contrast to the viruses as a whole, including plant viruses. They do not induce the production of interferon It does not lead to a serologic reaction. The pathogenicity of the viroids lays beyond the classical definition of the infectious diseases by Pasteur and Koch.
The immunogenous differences between the two types of pathogens have to do with the fact that viruses cause transcriptional diseases whereas viroids cause post transcriptional diseases.
Viruses primarily upset the synthesis of all proteins and encode instead their own virus proteins, which, in mammal organisms leads to an immediate immune response. Because of their relatively large size as particles and because of their protein and/or glycoprotein envelope, the classical viruses are the model for a potential antigen. Viral proteins are resented on the hista compatibility molecules of the class I (MCHI).
In contrast, viroids and viroid-analogous pathogens utilize exclusively the proteins of the hosts. This and their small size eliminate right from the start any presentation as antigen with the resulting immunogen reaction.
The metabolic damage effects of the viroids begins at a relatively late stage by post transcriptional disturbance of the protein and glycoprotein cycle of the host. Caused by the viroids, this may lead to:
This leads to concentration accumulations of nontransferred, nonrecycled and, depending on the circumstances, processed host proteins in stagnating cellular or intercellular pools where, by crystallization, mineralization, fibrillization, and/or amyloid plaque formation, they become immune to attacks by proteinases and are irreversibly deposited. The viroid-caused malfunction of the body's proteins permits the immune system only to employ autoimmune antibodies and this only at a late stage after the disturbance has become irreversible, that is, in the final stage of the disease. In fact, in the late lethal stages of subacute diseases caused by viroid-like particles in animals, autoimmune antibodies were found which are directed against the crystallized proteins of the host. With mice, an influence of the histacompatibility genes of the class II(MHCII) on the course of the infection has been found. The sponge-like perforation of infected nerve tissue as a result of inflammation-free spontaneous cell destruction reminds of apoptotic autodestruction mechanisms of the cells like they can be initiated by autoimmune signaling. By their primitivness and their sequentially genomic proximity as well as their compatibility to functional host cell ribonucleic acids (intranen, 7S, 7SL-RNA's, ribonucleasis P and L) viroidal and assimilated infectious particles have eluded up to now any selective curative influence since each directed action against the viroid parasite affects negatively also essential host cell functions.
Of all parasites the viroids and other subviral molecules have the closest functional proximity to the components of the host cell, from which right away maximal difficulties with regard to their identification and their control can be expected.
Viroids of plant cells can be isolated as single structures and are visible in an electron microscope. Blot methods are highly sensitive and permit the determination of a single viroid per cell.
In the animal and human area however, it has not been possible so far to observe and isolate disease-specific viroid-analogue ribonucleic acids in free form as unpackaged autonomically operating particles, but they were found in infected tissues. Under the physiological conditions of the animal cell, ribonucleic acid (RNA) is packaged in protein at the strands forming during the transcription, the replication and the processing. Specific bond affinities to molecules of the host cell resist the existence of naked RNA-viroid particles. In addition, the body temperatures present in warm blooded beings favor the dissolution of eventually possible autonomous structures of animal viroids. At 38.degree. C. also plant viroids are present in already molten modifications which are highly susceptible to associations.
The energies occurring with intermolecular associations are larger than those which can be obtained by the actuation of the still available intramolecular self complementary base airs (1,2 kcal/pair of nucleotides). The complexes are more stable than the viroid particle on its own. Viroid-like pathogens in animal cells are permanently in an ecliptic state. Their transmission by means of infected cell material does not require the formation of singly packaged visible particles as it is the case with viruses; this would be prevented already by the low coding capacity of such viroidal and other subviral RNA's.
Specific intermolecular bonds of subviral RNAs exist to host-cellular nucleonic acids (anti-sens-RNA-segments, anti-genomic bonds) to lipids (phospholipids) and to the enzymatic and physicochemical highly resistant fibrilous or amyloid pathogenic protein associates. The pathogens are packaged and masked. The association becomes biologically indestructible by reciprocal stabilization of its components.
Fibrilous and amyloid protein aggregates form stagnating pools presumably with the cooperation of the viroidal parasites. This leads among others to post-transcriptional point imitations of host-coded normally not pathogenic proteins; In certain positions hydrophilic amino acids are substituted by lipophilic and acidic ones by basic amino acids. The bundled pathogenic, highly resistant, prion-protein aggregates are believed by some authors to have the capability to replicate.
In parallel therewith, the afflicted neurons have an increased affinity to basic microscopic coloring agents (basophilic character).
A strong lipophilic affinity of the pathogens or their associations contributes to their capability to undermine defense reactions from the aqueous serological environment. The shifting to the lipophilic environment is also apparent from the preference of the pathogens for lipid-rich tissue in the invaded host: the brain, the nerve strings, the spinal cord, the hypophysis, the spleen, the eyes, the lymphocytes, the intestinal tract and all secretarial tissues are main target organs.
Pathogens in such infectious tissues can be inactivated by treatment with lipophilic solvents (acetone). Plaques and fibrils of the prion-proteins can be dissolved by subsequent exposure to nucleases and proteinase K.
While the replication cycle of the viroids in plant cells is known, the activity of viroid-like particles in the animal and human areas is not yet understood.
It is known, however, that all types of viroids are highly infectious and that the viroidal particles are quasi-indestructible. In contrast to bacteria and viruses, it is practically impossible to eliminate them from a contaminated environment and this leads to a cumulative biocontamination.
For this reason, it is economically highly important that viroid infections in the plant and animal area can be controlled without destructive side effects.
In the plant environment, viroid infections lead to enormous damage over large areas, particularly in tropical climate zones. Wild and cultured plants are equally affected in those areas.
The coconut Cadang cadang Viroid (CCCVd) has destroyed already 30 million coco palm trees (Cocus nucifera). The ultra small particles of 10 nm cause 20 m high tree giants to die, 800,000 per year in the Philippines. The destruction in herd-like expanding zones is so complete and the transmission of the viroids so unavoidable and prompt, that extinction of the respective species and of the viroid-tolerant related species must be feared.
In moderate climate areas, selective monocultures and green house plants have experienced heavy viroid epidemics, particularly high performance species which were grown for particular properties. Wild plants barely develop symptoms, but they provide a permanent reservoir for new epidemics in the cultured plants.
One of the most virulent viroids in our climate zone is the spindle tuber viroid (PSTVd) of the potatoes. It is polyvalent and attacks a large number of cultured plants. With 356 to 360 ribonucleotides, it belongs to the large viroids (The upper existence limit for the viroids known so far is about 375 ribonucleotides).
Large viroids such as PSTVd have a large host circle. Small viroids such as the above mentioned CCCVd (246 ribonucleotides) attack only one or a few related species. In this connection, it is to be pointed out that the known viroids are not evenly distributed over the narrow range of existence between 246 and 375 ribonucleotides, but that they are present in a cumulative manner: At the upper and lower limits and in between at distances of about 20 nucleotides, there is an accumulation of the various viroid types. The nucleic numbers in between are unoccupied.
PSVTd can occur in isolates with mild, strong or lethal symptom development. Field potato cultures infected with strong isolates suffer yield losses in warm summers of 60% (growth delay, small fruits). Greenhouse plants are even more susceptible. A virulent PSTVd strain can eliminate greenhouse plants 100%.
In the animal and human area viroid-like particles and viroid segments integrated in viral genomes are suspected to be responsible for a number of degenerative non- or post inflammatory diseases of the central nervous system. After long incubation periods and latent periods, they can lead to grave neuropathologic disturbances and then always to death.
Viroid-like parasites are believed to be responsible in mammals and in humans solely or as a cofactor for about 50 subacute chronic illnesses. They are listed in the following 5 groups in the order of lower confirmation of the knowledge concerning the viroid participation:
The importance of the non-conventional viruses is increased by the suspicion that they are co-responsible for a long list of other chronic diseases; Alzheimer's dementia (clinically not distinguishable from a slow CTE, 800,000 potential cases in Germany which occur at increasingly younger ages), the Parkinson's disease, numerous autoimmune diseases, sclerosis, for example, lateral sclerosis, epilepsies, schizophrenias, endogene depressions, behavior anomalies and the Autism.
Retroviruses and viroids have a number of common properties: Classic viruses cause inflammatory processes, viroids cause degenerative processes. Retroviruses combine both properties and functionally bridge the difference between the classical viruses and the viroids.
Viroids, on the other hand, have retroviral properties. Because of their ability to become an integral part of a genome and again to splice out therefrom, they are, on an RNA basis, the functional equivalent of the retroviral proviruses in the DNA genome with whose long terminal repeats (LTR), there are sequence homologies. Such noncoding sequencing repetitions play a role in the translation or retranslation of viral information between RNA and DNA. Highly conserved viroid sequences, which are present in all viroids, appear to be active also in retroviral genomes.
There are analogies also for the replication cycle: Retroviruses and viroids use the same cellular enzymes: DNA-dependent RNA polymerases.
A controlled modification of the pathogenic properties of the relatively simple viroids may contribute to a better understanding of the role of the LTR in the much more complex retroviruses and to a controlling influence on the latter.
Furthermore, the genomes of the DNA viruses of the families Parvor, Hepadena, Polyoma, Adeno and Herpeto also have sequence repetitions which contain the typically conserved viroid sequences as basic building block. With their multiply repetitious "Rolling Circle" mechanism, the viroids are the base model for the formation and functioning of noncoding sequence repetitions on the basis of RNA and DNA.
Generally, it seems that conserved viroid sequences (for example, the central palindromic deka nucleotides of the viroids) are necessary for the interaction of the viroids between the active replication phase and the occasional disappearance in the DNA genome of a host which shows no symptoms during that period.
A viroids participation in these late follow-up infections is under discussion for two reasons:
On the other hand, double strand RNA segments act as pivots for the stabilization of RNA virus genomes and, in this manner, are responsible for the distribution width of the quasi-species population and the amount of the defective subspecies formed. The more remote such defective subspecies are from the initial antigen specimen, the better are their chances for survival.
Viroids and assimilated infectious particles have an unusual physical, chemical and biological resistance.
The inactivation by UV, ionizing and neutron irradiation, by heat, formaldehyde and alcohol and by nucleases and proteins is effective only in a limited manner.
The potato Spindle Tuber viroid is 20.times. more resistant against UV irradiation than the most stable viruses, Scrapie-Agens are even 200.times. more resistant. The latter is also resistant to stomach acidity and temperatures of 350.degree. C. Land grazed on by Scrapie-infected sheep remains infectious for some years. The failure of the classical decontamination and disinfecting methods have been demonstrated in the human and animal medicine areas by spectacular accidents.
Viroids from diseased culture plants deposit themselves in wild plants as latent viroids or trace viroids, without causing any symptoms and they wait there for adequate hosts. They establish themselves firmly in a practical biological living area and survive the changes thereof.
By industrial farming with large area use of cultivating and cutting machinery, the active and latent viroid reservoirs are regularly and massively mixed. The vector density and velocity of the viroid transmission are therefore very high. Multiinfections (viroids, plant viruses) occur more and more frequently. No adequate defense mechanisms exist for this situation introduced by industrialization in contrast to the normal evolution and in an abrupt manner. The results are a large area destruction of sensitive (viroid tolerant) vegetation species and a cumulative bio contamination of the plant life by more and more viroid types and strains.
Also in the animal raising and human areas, the risk factors for a systematic transfection of the population by the pathogens of the subacute encephalopathies which came in contact with viroid like RNA molecules are increasing.
Keeping animals in large numbers under industrial conditions and the recirculation of waste from meat processing plants for the purpose of feeding have lead, among all breeding and domestic animals, to an expansion of Scrapie, cattle madness BSE and its variants. Among the same type of animals living in the wild, such epidemics are not known. At least sporadic infections pass without symptoms. If the same animals are subjected to the same conditions as domestic animals or if they are contained in ecological gardens, they also become ill.
The various pathogens overcome the species barrier between the mammals more or less easily, possibly by way of an intermediate host as they may be without symptoms for some time, like the plant viroids. Series paths in the new host then shorten the incubation periods and increase the virulence,
The use, since 1981, of ground feed of Scrapie infected sheep led in England in 1987 to an outbreak of cattle madness (BSE) which was unknown before, but is epidemic in England today and has worldwide sporadic outbreaks. All livestock and all domestic animals can be infected. Industrial growing methods, immunization and doping guns are potential causes for spreading infections.
It appears that for the various pathogen isolates, there is a correlation between the ease with which they overcome the species barrier and adapt to a new host and effectiveness with which they extinguish the infected individuals of the infected population: The not very easily adaptable species-stable Scrapie pathogen of the sheep cannot be easily transferred to other animal species, but completely destroys its host species, the infected sheep herd. On the other hand, the bovine BSE pathogen, which easily switches from one to another species, leads to fatalities in only a few individuals of the infected cattle herd. That means that, in a relatively unspecific pathogen strain (with a wide host spectrum), the infected individual has a better chance to delay or avoid development of lethal symptoms.
In order to estimate the potential risk for humans, man must also be considered to be a mammal living in a concentrated life style.
A transfer between animal and human, for example by contaminated live stock and domestic animals, has occurred epidemiologically by Scrapie (sheep) Creutzfeldt-Jakob-Encephalopathy (human) and it has been experimentally shown that humans can be a vector and infect animals. The earlier described target organs of the pathogens were particularly infectious: The nerves, lipid and secretarial tissues, (the sale of which is no longer permitted in France if they originate from cattle or eggs). But also muscle tissue and milk products can not be excluded from the contamination chain.
The human to human transfer of the spongiform encephalopathies in the area of medicine has been proved by dramatic special cases. The epidemic extent cannot yet be judged, because the pathogen appeared only recently and the incubation period is so long. Preventative measures are hindered by:
A post infectional treatment has not been known so far either.
With the present state of the art, a potential risk of a transmission is present:
Consequently, all the negative conditions for a cumulative bio-contamination by viroids of the higher plants as well as of the higher animals by viroid-like particles are in place.
The fight of the smallest against the largest occurs in the metabolism with minimal differences. The lack of information and incapability of coding of the pathogens have the effect that they proliferate exclusively on the basis of host factors. With their expansions across individuals and across species, the virulence of the viroid pathogens increases (in contrast to viruses where the species transfer may result in less virulent viruses).
The industrialization of the nature increases the susceptibility of the hosts: Particularly overbred animals and plants grown in large quantities and in monocultures are highly susceptible.
As a result, isolated mutations and statistically rare cases of faulty processing of genetic products, which may occur sporadically in individuals could lead to a global pandemic whereas, with the undercritical conditions existent in the pre-ndustrial society, they would have been limited to island-like areas and would have self-destructed.
A miniature model for a global ecological problem situation of the "slow virus" epidemic is the Kuru disease in New Guinea which can safely be traced to a sporadic case of Creutzfeld-Jakob (CJE) of a Papua aborigin. The ritual buryial cannibalism became the vector of the Kuru epidemic which extended also to uninvolved small children who were contaminated only by accidental contact with the infectious tissue. In 1957, the buryial ritual was no longer permitted by law. This eliminated the main vector. As a result, the epidemic subsided. Especially, no children born afterwards became sick. But even today, Papuan adults anywhere in the world may die as a result of a participation in a buryial ritual that happened more than 30 years earlier.
Altogether in a number of years, a sum of risk factors became apparent; dangerous, infectious, highly resistant, ubiquitary, ultra small pathogens with extremely long incubation periods and metabolic minimum differences from host factors are contrasted by less and less resistant host organisms.
In the animal and human areas, it is further to be considered that the pathogens are undetectable, that is, they are transmitted in various ways, that there are no adequate tests and that there are no specific disease symptoms so that a certain diagnosis can be obtained only after death.
An antidote must be capable of distinguishing in a highly specific manner between the normal and the quasi-identical pathogenic molecules on both sides, the host and the pathogenic side and must be able to selectively affect the latter ones in an inhibitory manner.
Such an antidote against viroids and viroid-like pathogens has not been known so far with the exception of cell poisons.
It is the object of the invention to show a new use of boron compounds which can be bio-assimilated.